Oxidative stress remains a central causative factor in the etiology and progression of myriad vascular and non-vascular diseases, including among many others cardiovascular diseases, cancer, neurological disorders, and other pathologies. A major source of reactive oxygen species (ROS) is a family of enzymes, NADPH oxidases (Nox), that catalyze electron transfer from NADPH to molecular oxygen to give ROS, such as superoxide (O2.−) and/or hydrogen peroxide (H2O2). Nox plays a crucial role in signaling cascades initiated by pro-inflammatory stimuli including hormones, vasoactive agents, and cytokines as well as mechanical stress. Members of this family include Nox1-5 as well as Duox1 and 2; in the human cardiovascular system, Nox1, 2, 4, and 5 isoforms are prevalent.
The major catalytic subunit of these Nox isozymes possesses six transmembrane domains with a cytosolic C-terminus containing NADPH- and FAD-binding domains. Specifically, Nox1, 2, and 4 are constitutively associated with membrane-bound p22phox, the complex of which forms cytochrome b558. On the other hand, Nox5 does not require p22phox or cytosolic subunits but uniquely contains calcium-activating EF domains at its N-terminus. Furthermore, the Nox isozymes differ in requirements for specific cytosolic subunits for activation and organization. Nox1 associates with GTPase Rac1, cytosolic activator NoxA1, and cytosolic organizer NoxO1. Nox2 associates with Rac1 or Rac2 as well as cytosolic activator p67phox and cytosolic organizer p47phox while Nox4 requires no classical cytosolic subunits but is regulated by Poldip2 The result of activation of these enzymes is the generation of ROS in the form of O2.− (Nox1, 2, 5) and H2O2 (Nox4). ROS production is mediated by electron transfer from NADPH in the cytosol to FAD to form FADH2. Single electron transfer to heme groups on the transmembrane domains and subsequent transfer to molecular oxygen on the opposite side of the membrane forms O2.−, which can be converted to H2O2 by superoxide dismutase (SOD). Downstream effects of this ROS generation include changes in gene expression, cellular signaling, host defense and inflammation, and cell growth regulation. The inability of currently available agents to specifically inhibit a particular NADPH oxidase along with the combinative and varied expression of these isozymes in cells and tissue has made it difficult to assess their individual contributions to disease. Additionally, due to the wide distribution of the Nox enzymes in a variety of cells in the body as well as their beneficial role in signaling, nonspecific Nox inhibitors are likely to cause undesired effects in vivo.
Among the isoforms, Nox2 (aka gp91phox, the first Nox isoform discovered) has been implicated in cardiovascular disease (CVD) processes including atherosclerosis, hypertension, ischemia reperfusion, cardiac hypertrophy, cardiomyopathy, stroke, and restenosis. In addition to CVDs, Nox2 has more recently been implicated in neurodegenerative diseases such as Huntington's, Alzheimer's, and Parkinson's diseases. Small molecules are the preferred therapeutic strategy for clinical use. However, due to the complex assembly and the high degree of homology among the various members of the Nox family, the development of isoform-specific inhibitors has proven challenging.